One of the major problems today is defining the control mechanisms involved in regulation of the cell cycle. A number of protein kinases have been shown to be involved in activation of the cell cycle, both at the hormonal level and during progression through the cell cycle. Our studies have focused on a unique protein kinase, PAK I, which is involved in maintaining cells in a non-dividing state. In the proposed studies we will continue characterization of the protein kinase and examine the role of PAK I in regulation of the cell cycle. This protein kinase appears to be of considerable importance in cell cycle regulation. A. Complete the cloning, sequencing and analysis of the cDNA for PAK I. Quantify mRNA levels for PAK I in cells and tissues. Express recombinant PAK I in E. coli and/or insect cells. B. Determine the recognition sequence for PAK I with synthetic peptides and identify protein substrates for PAK I. Examine the effects of phosphorylation on regulation of substrate activity. Site-specific mutations of PAK I phosphorylation sites in proteins shown to be regulated by PAK I will be prepared and analyzed for changes in activity. C. Analyze the role of PAK I in regulation of the cell cycle utilizing mammalian cells. Characterize the active form of PAK I in 3T3-L1 cells and examine the requirements for activation in vivo. D. Identify the protein kinases which phosphorylate PAK I in vitro and the sites of phosphorylation. Examine the effects of phosphorylation and autophosphorylation on the activity of the purified enzyme. Site-specific mutations of the phosphorylation sites on PAK I will be prepared and the effects of these mutations on PAK I activity will be analyzed. Results from the proposed research will enable us to complete the characterization of PAK I, to analyze the role of PAK I in maintaining somatic cells in a non-substrates involved in growth control and to examine the regulation of PAK I activity.